ADP-ribosylation factors (ARFs) are a family of about 20-kDa guanine nucleotide-binding proteins originally identified and purified based on their ability to enhance the ADP-ribosyltransferase activity of cholera toxin. More recently, ARFs have been identified as critical participants in vesicular trafficking pathways and activators of a phospholipase D. A 64-kDa protein with an 18-kDa carboxy-terminal ARF domain and a 46-kDa amino-terminal ARD1, was initially identified by cDNA cloning. Using recombinant fusion proteins, we have shown that the amino-terminal domain of ARD1 (p5) controls the activity of the domain (p3) by stimulating its intrinsic GTPase activity. We have now confirmed that ARD1 is a member of the guanine nucleotide-binding protein family and defined some characteristics of its unusual GTPase activity. We found that for either ARD1 or its ARF domain (p3), cardiolipin promoted both GTPgammaS binding and CTA activation more effectively than did the other phospholipids tested. Using chimeric proteins and site-directed mutagenesis, we showed that ARF1, which is not substrate for the GTPase-activating domain of ARD1 (p5) and does not physically interact with it, acquired both characteristics after Gly 40 and Thr 45 were replaced with, respectively, Asp and Pro. The amino acids Asp 25 and Pro 30 of the ARF-domain (p3) of ARD1 were, therefore identified as participants in functional and physical interaction with the GTPase-activating domain of ARD1. Finally we demonstrated that the carboxy-terminal part of p5 stabilized the ARF- domain of ARD1 in a GDP-bound state, i.e., acted as a GDP-dissociation inhibitor. These results are consistent with the view that ARD1 is a unique member of the monomeric G protein family as it, like the alpha- subunits of hetero trimeric G proteins possesses its own GTP hydrolysis- activating domain.